The Rho family of G proteins regulate diverse cellular processes involving coordinate alterations in cytoskeletal rearrangements and transcriptional control, and ultimately are necessary for processes as varied as chemotaxis, phagocytosis, and cellular growth and differentiation. Conversely the aberrant functioning of Rho-family G proteins promotes various malignancies from irregular development to cellular transformation and oncogenesis. A large and diverse class of guanine nucleotide exchange factors related to Db1 control the regulated activation of Rho-family G proteins by facilitating the release of GDP bound to inactive G proteins and catalyzing the concomitant loading of GTP to produce G proteins active in downstream signaling. Consequently, dysfunctional constitutive activation of Dbl-related GEFs abnormally elevates intracellular concentrations of active, GTP-bound G proteins contributing to tumorigenesis and developmental disorders. Although GEFs specific for Rho-family G proteins vary greatly in size and domain architecture, all contain a Db1-homology domain (DH) invariantly associated with a juxtaposed, C-terminal pleckstrin homology domain (PH). DH domains catalyze guanine nucleotide exchange, while functions for the associated PH domains are less well defined and more variable. This proposal describes experiments designed to illuminate, at atomic resolution, the conserved mechanism of guanine nucleotide exchange catalyzed by Dbl-related GEFs. We have recently solved the crystal structures of the DH/PH fragments of Tiam1 and Dbs in complex with their cognate G proteins, Rac1 and Cdc42, respectively. These structures form the basis for understanding guanine nucleotide exchange catalyzed by Dbl-related GEFs and guide many of the biophysical, biochemical, and in vivo experiments described. Complementary aspects of this proposal are designed to elucidate mechanistic details required for efficient guanine nucleotide exchange catalyzed by Dbl-related GEFs, including assigning specific functions to PH domains invariantly associated with DH domains. It is anticipated that this information will be invaluable for understanding and controlling the activation of Rho-family G proteins culminating in the amelioration of pathologies associated with constitutively active G proteins.